Effects of Progesterone on the Development of the Rat Morula*

Vol. 21, No.7, July 1970 Printed in U.S.A.

FERTILITY AND STERILITY

Copyright

© 1970 by The Williams & Wilkins Co.

EFFECTS OF PROGESTERONE ON THE DEVELOPMENT OF THE RAT MORULA * ZEEV DICKMANN, PH.D. Departments of Gynecology and Obstetrics and Anatomy, University of Kansas Medical Center, Kansas City, Kansas

We have considerable knowledge about the effects of sex hormones on egg transport,9 sperm transport,2 capacitation,! and fertilization. s Less information is available about the effects of these hormones on the development of the fertilized egg. s The aim of the present study was to determine the effects of progesterone on the development of the morula in rat. The period of morula-blastocyst transformation may be highly sensitive to environmental changes, because during this period the cells of the embryo undergo differentiation for the first time. 11, 12 It was, therefore, of interest to determine whether deviation from the normal estrogen/progesterone conditions would affect the development of the morula. MATERIALS AND METHODS

Young adult, virgin female rats of the Holtzman strain weighing 180-240 gm. were used. Eggs were recovered from donors and transferred into the uteri of recipients, according to the method of Dickmann and De Feo. 8 Donors of morulae (experiments I-X, XII, and XIII) were Day 4 pregnant females (Day 1 was indicated by spermatozoa in vagina), and donors of blastocysts (Experiment XI) were Day 5 pregnant females. The recipients were divided into 13 treatment groups. The treatment common to all of the recipients was as follows. On the day of proestrus (Day 0) and again on the following day (Day 1), recipients

* Supported in part by the Population Council and the Ford Foundation.

were cervically stimulated with a vibrating rod to induce pseudopregnancy,4 and on day 2 they were ovariectomized. Eight to 10 morulae (or blastocysts) were transferred into each recipeint. Progesterone given alone, estrone given alone, and estrone plus progesterone given together, were suspended in 0.1 ml. of sesame oil and injected subcutaneously {for dosages, see Fig. 1 and Table 1). The inj ections were administered between 08.00 and 09.00 hr. Ovariectomy, egg transfer, and killing of recipients were done between 09.00 and 11.00 hr. Experiment I. Recipients were treated according to the schedule shown in Fig. 1. At the end of the experiments, the recipients were killed, and the number of fetuses and implantation sites were recorded. It should be noted that the schedule of Experiment I was formulated after the schedule often used for the experimental induction of delay of implantation (i.e., ovariectomy on Day 2 of pregnancy followed by daily inj ections of progesterone. Under these conditions, morulae enter the uterus on Day 4). Thus it was anticipated that an appreciable percentage of the transferred morulae would develop into fetuses. Experiments II-X. The schedules of these experiments differed from that of Experiment I in that the period between ovariectomy and morulae transfer was lengthened. During this extended time, the recipients received various treatments shown in Table 1. The treatment which followed morula transfer was the same as

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542

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MORULA TRANSFER

I

3

I I

4

I

5

CERVICAL STIMULATIONS

in Experiment I (Day 5-21, Fig. 1), namely, 2 days of progesterone, followed by 14 days of estrone plus progesterone, followed by killing the recipients. Experiment XI. The schedule of this experiment was the same as that of' Experiment V (Table 1), but, instead of morulae, blastocysts were transferred. Experiments XII and XIII. The schedule of these experiments was the same as that of Experiment V, except that the recipients were killed 24 and 72 hr., respectively, after transfer, at which time the eggs were recovered and examined in a drop of 0.9% NaCl solution under a compound microscope. RESULTS AND COMMENTS

Experiments I-V. The results, summarized in Table 1, show that, when morula transfer was preceded by 2, 3, 4, 5, and 6 days of progesterone treat~ent, 49% (Experiment I), 38% (Experiment II), 13% (Experiment III), 2.5% (Experiment IV), and 2% (Experiment V) of the morulae developed into fetuses. These results can be interpreted in three ways. (1) Overstimulation with progesterone interfered with embryonic development. (2)

KILL

Lack of estrogen interfered with embryonic development. Lack of estrogen is considered here in terms of lack of estrogen effect on target organs, and not just in terms of lack in the circulation. Although the recipients were deprived of their estrogen source by ovariectomizing them, an estrogen effect very likely still remained for some time after ovariectomy.5 As this time increased, the estrogen effect progressively diminished, and by Days 7 and 8 (Table 1) the estrogen effect reached a low point no longer compatible with survival of the embryo. (3) The interference of embryonic development was caused by the factors indicated in both 1 and 2. Experiments VI-VIII. In these experiments, as in Experiments I-V, lengthening the period of progesterone treatment before morula transfer progressively increased embryonic loss (Table 1). Because the time from ovariectomy (i.e., removal of the estrogen source) to transfer was the same in all three experiments, these results tend to favor the interpretation that progesterone overstimulation, and not lack of estrogen, was the primary cause for the interference with embryonic de-

July 1970 TABLE

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PROGESTERONE AND RAT MORULA

1. The No. of Fetuses and Implantation Sites a Resulting from the Transfer of Morulae into the Uteri of Recipients Receiving V arious Hormonal Treatments Trea tmen t on daysb recipients

morulae

transferred

No. and % of fetuses

No. and % of implantation sites

9

78

38 (49 )

23 (29 )

11

84

32 (38)

22 (26 )

12

112

15 (13 )

23 (21 )

13

120

3 (2.5 )

24 (21 )

15

136

3 (2)

18 (13 )

12

111

49 (44)

24 (22)

15

126

27 (21)

36 (29 )

14

123

16

145

60 (41 )

25 (17 )

13

122

12 (10)

32 (26 )

No. of

Experiment 2

3

4

5

6

-- ------ -TRd 0pe p p TR II 0 P P P P TR III 0 P P P P P IV 0 P P P P P 0 V P P P P P VI 0 NT! NT NT NT P 0 VII NT NT NT P P VIII 0 NT P NT P P IX 0 Eu NT E P P X 0 P P P E E

7 8 ----

I

TR

No. of

P TR

P

P TR

P

P TR

P

P TR

P

TR

P

19 (15.5 )

P TR

P

8 (6.5 )

P

P

A site at which a blastocyst implanted, but where sometime later the embryo died and was resorbed. In Experiments I-X, the preovariectomy and the post-transfer treatments were as is shown in Fig. 1. - 0, ovariectomy. d TR, transfer of morulae . • P, progesterone, 2 mg./day. ! NT, no treatment . • E, injections of a divided dose of 2 p.g estrone/day; 1 p.g at 08.00 and 1 p.g at 16.00 hr.

a

b

velopment. Nevertheless, the possibility was considered that estrogen might antagonize the action of progesterone and thereby reduce embryonic loss. This possibility was explored in the next set of experiments. Experiments IX and X. The results of these two experiments (Table 1) confirmed again that, the longer the pretransfer progesterone treatment, the higher was the mortality of embryos. In Experiment IX, in which 3 days of progesterone treatment preceded transfer, 41% of the transferred morulae developed into fetuses; by contrast, in Experiment X, in which 4 days of progesterone preceded transfer, only 10% of the transferred morulae developed into fetuses. Thus 1 additional day of progesterone pretreatment reduced em-

bryonic survival to about one-fourth of the number surviving in Experiment IX. If we compare 3 vs. 4 days of progesterone pretreatment in the previous two sets of experiments, we note that the 4-day pretreatment reduced embryonic survival to about one-third:' 38% (Experiment II) vs. 13% (Experiment III), and 21% (Experiment VII) vs. 6.5% (Experiment VIII). Comparing the yield of Experiment IX (41 %) (estrone-treated) with that of Experiment VII (21 %) (not estrone-treated) shows that the estrone treatment reduced considerably the embryonic mortality when transfers were preceded by 3 days of progesterone treatment. On the other hand, when transfers were preceded by 4

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days of progesterone treatment, the estrone treatment used had only a slight effect on embryonic survival: 10% (Experiment X, estrone-treated) vs. 6.5% (Experiment VIII, not estrone-treated). Conclusions jor Experiments I-X. Based on the results obtained, it is concluded that overstimulation with progesterone interferes with embryonic development and that, under certain conditions, the deleterious effects of progesterone can be reduced or even prevented by pretreatment with estrogen. The next question asked was: at what stage of development was the embryo damaged by the progesterone treatment? Experiment XI answered this question. Experiment XI. The schedule of this experiment was the same as that of Experiment V (Table 1) with one exception-instead of morulae, blastocysts were transferred. Eighty-eight blastocysts were transferred into the uteri of 11 recipients. The transfers yielded 55 (62.5%) fetuses and 6 (7%) .implantation sites. Since in the same type of recipient, blasto cysts yielded 62.5% fetuses whereas morulae (Experiment V) yielded 2% only, it is obvious that the injurious effect of progesterone was exerted during the period when transformation from morula to blastocyst should have occurred. The last two experiments (XII and XIII) explored the type of damage caused to the embryo by progesterone overstimulation. Instead of testing for formation of fetuses, the fate of the morulae was studied at 24 and 72 hr. after transfer. Experiment XII. The schedule of this experiment was the same as that of Experiment V (Table 1) from Day 0 to Day 9. However, on Day 9, the recipients did not receive progesterone; they were killed, and their uteri were excised and flushtd to recover the eggs which were examined in 1 drop of 0.9% N aCI solution under a compound microscope.

Eighty-one morulae were transferred into the uteri of 8 recipients. Twenty-four hours later, 64 of the 81 eggs (79%) were recovered. They were classified in three categories: morulae (eggs which looked like morulae and did not show signs of degeneration), blastocysts (eggs which looked either normal or abnormal, but had a blastocoele, and did not show signs of degeneration), and degenerated eggs. Following this classification, of the 64 eggs recovered, 22 (34%) were morulae, 14 (22%) were blastocysts, and 28 (34%) were degenerated. An example of each category of eggs is shown in Figs. 2-6. Experiment XIII. The schedule of this experiment was the same as that of Experiment V from Day 0 to Day 11. However, on Day 11, the recipients were not inj ected with estrone plus progesterone; instead they were killed and the eggs were recovered and examined as in Experiment XII. One hundred twenty-seven morulae were transferred into the uteri of 12 recipients. Seventy-two hours later, 88 of the 127 eggs (69%) were recovered: 3 (3%) were morulae, 8 (9%) were blastocysts, and 77 (87.5%) were degenerated eggs (Figs. 712) .

In Experiment V, the yield was 2% fetuses plus 13% implantation sites-a total of 15%. Based on this result, we can conclude that in the present experiment the morulae and blastocysts, representing 12% of the recovered eggs, had the potential to form implantation sites, and a fraction of them to develop into fetuses. DISCUSSION

In the rat, the transformation of morula to blastocyst occurs between Days 4 and 5 of pregnancy, at which time the female is' under the influence of estrogen and progesterone. A basic question is whether or not these hormones influence morula-blastocyst transformation. In a recent study it

The types of eggs recovered from recipients in Experiment XII. (For further explanations see text.)

X 515. FIG. 2. Morula FIG. 3. Blastocyst with small blastocoele (light area at 3 o'clock) FIG. 4. Blastocyst FIG. 5. Degenerating egg. The general outline of a morula is still recognizable FIG. 6. Completely degenerated egg

545

The types of eggs recovered from recipients in Experiment XIII. (For further explanations see text.) X 515. FIG. 7. Morula. Of the three morulae recovered, this was the best looking one FIG. 8. Blastocyst. Of the eight blastocysts recovered, this was the best looking one FIG. 9. Blastocyst with small blastocoele (at about 6 o'clock) FIG. 10. Blastocyst with abnormal blastocoele formation FIG. 11. Egg showing early fragmentation and degeneration FIG. 12. Completely degenerated egg

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rROGESTERONE AND RAT MORULA

was shown that morulae developed into normal blastocysts subsequent to their transfer into the uteri of long term ovariectomized recipients, demonstrating that the ovarian hormones are not necessary for transformation. 6 The fact that transformation can occur . in the absence of the ovarian hormones does not necessarily mean that these hormones cannot influence the development of the morula. Indeed, the present results have shown that morulae degenerated in the uteri of reciepients which had been ovariectomized and inj ected daily with a physiologic dose (2 mg.) of progesterone. The results further suggest that estrogen can antagonize the deleterious effects of progesterone. It is therefore concluded that when estrogen and progesterone are present they have to be in proper balance; otherwise pregnancy will be terminated. Overstimulation with progesterone causes degeneration of the morula and overstimulation with estrogen causes expulsion of the eggs from the reproductive tract.7, 10 In the rabbit, too, progesterone caused degeneration of fertilized eggs in vivo. 3 Whitten 13 has shown that 8-cell mouse embryos grown in vitro degenerated when progesterone, in concentrations of 4 p.g./ ml. or higher, was added to the culture medium. The results of the latter experiment show that progesterone can have a direct toxic effect on fertilized eggs. However, Whitten's results do not constitute proof that when inj ected into the animal progesterone acts directly on the embryo. It, therefore, remains to be determined whether, in the present study and in Chang's3 study, progesterone acted on the embryo directly or whether it acted on the uterus whose secretion, in turn, affected the embryo. SUMMARY

The egg transfer technic was used to study the effect of progesterone on the survival of rat morulae in vivo. Morulae

were transferred into the uteri of recipients which had been ovariectomized on Day 2 of pseudopregnancy and inj ected with progesterone for a varying number of days. After transfer the recipients were injected with progesterone for 2 additional days and progesterone and estrone for 14 days: the recipients were killed and examined for presence of fetuses. When morula transfer was preceded by 2, 3, 4, 5, and 6 days of progesterone treatment, 49%, 38%, 13%, 2.5%, and 2%, respectively, of the morulae developed into fetuses. It was shown that the deleterious effects of progesterone can be reduced by pretreatment with estrone. It was proved that progesterone is deleterious to the morula, but not to the blastocyst. Acknowledgments. The technical assistance of J. R. Hart and the typing of the manuscript by Judy Ash are gratefully acknowledged. REFERENCES 1. BEDFORD, J. M. Experimental requirement for capacitation and observations on ultra-structural changes in rabbit spermatozoa during fertilization. J Reprod Fertil (Suppl. 2) :35, 1967. 2. BISHOP, D. W. "Biology of Spermatozoa." In Sex and Internal Secretions (Vol. 2), Young, W. C., Ed. Williams & Wilkins, Baltimore, 1961. 3. CHANG, M. C. Fertilization, transportation and degeneration of eggs in pseudopregnant or progesterone-treated rabbit. Endocrinology 84: 356, 1969. 4. DE FEO, V. J. Vaginal-cervical vibration: A simple and effective method for the induction of pseudopregnancy in the rat. Endocrinology 79 :440, 1966. 5. DICKMANN, Z. Hormonal requirements for the survival of blastocysts in the uterus of the rat. J Endocr 37 :445, 1967. 6. DICKMANN, Z. Hormonal requirements for the transformation of morula to blastocyst in the rat: Effects of long-term ovariectomy. Steroids 14:385,1969. 7. DICKMANN, Z. Unpublished data. 8. DICKMANN, Z., AND DE FED, V. J. The rat blastocyst during normal pregnancy and during delayed implantation, including an observation on the shedding of the zona pellucida. J Reprod Fertil13 :3, 1967.

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9. GREENWALD, G. S. "Hormonal Regulation of Egg Transport through the Mammalian Oviduct." In Progress in Fertility, Behrman, S. J., and Kistner, R. W., Eds. Little, Boston, 1968.

10. HADDAD, V., AND KETCHEL, M. M. Termination of pregnancy and occurrence of abnormalities following estrone administration during early pregnancy. Int J FertiI14:56, 1969. 11. MINTZ, B. Formation of genetically mosaic

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mouse embryos, and early development of "lethal (t12/t'2)-normal" mosaics. J Exp Zool 157 :273, 1964. 12. TARKOWSKI, A. K. "Discussion." In Preim-

plantation Stages of Pregnancy Ciba Found. Symp., Wolstenholme, G. E. W., and O'Connor, M., Eds. Churchill, London, 1965, p. 138. 13. WHITTEN, W. K. The effect of progesterone on the development of mouse eggs in vitro. J Endocr 16 :80,1957.